A new tool to reveal structure of proteins

For roughly a decade, a technique called solid state nuclear magnetic resonance (NMR) spectroscopy has allowed researchers to detect the arrangements of atoms in proteins that defy study by traditional laboratory tools such as X-ray crystallography. But translating solid state NMR data into an actual 3D protein structures has always been difficult.

In the current online edition of Nature Chemistry, Christopher Jaroniec, associate professor of chemistry at Ohio State University, and his colleagues describe a new solid state NMR method that uses paramagnetic tags to help visualize the shape of protein molecules.

"Structural information about biological molecules is critical to understanding their function," Jaroniec said. "Our new method promises to be a valuable addition to the NMR toolbox for rapidly determining the structures of protein systems which defy analysis with other techniques."

Such protein systems include amyloids, which are fibrous clusters of proteins found in diseased cells, and associated with the development of certain neurological diseases in humans.

"Although for the purposes of the paper we tested the method on a small model protein, the applications are actually quite general," Jaroniec added. "We expect that the method will work on many larger and more challenging proteins."

Protein molecules are made up of long chains of amino acids folded and wrapped around themselves, like tangled spaghetti. Every type of protein folds into its own unique pattern, and the pattern determines its function in the body. Understanding why a protein folds the way it does could give scientists clues on how to destroy a protein, or alter its function.

To test their method, the researchers chose a protein called GB1, a common protein found in Streptococcus bacteria. GB1 has been much studied by scientists, so the structure is already known. They engineered a form of the protein in which certain amino acids along the chain were replaced with a different amino acid – cysteine – and created the right chemical conditions for yet another tag – one containing an atom of copper – to stick to the cysteine. The amino acid-copper tags are known as "paramagnetic" molecules, and they significantly influence the signals emitted by the different protein atoms in the magnetic field of an NMR instrument.

The researchers were able to determine the locations of the protein atoms relative to the paramagnetic tags, and use this information to calculate the folded shape of the GB1 protein.

Provided by The Ohio State University (news : web)

Scientists discover a surprising new way that protons can move among molecules

Hydrogen bonds are found everywhere in chemistry and biology and are critical in DNA and RNA, where they bond the base pairs that encode genes and map protein structures. Recently a team of researchers using the Advanced Light Source (ALS) at the U.S. Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) discovered to their surprise that in special cases protons can find ways to transfer even when hydrogen bonds are blocked. The team's results appear in Nature Chemistry.

Stacking the odd molecules

A group led by Musahid Ahmed, a senior scientists in Berkeley Lab's Chemical Sciences Division (CSD), has long collaborated with a theoretical research group at the University of Southern California (USC) headed by Anna Krylov. In recent work to understand how bases are bonded in staircase-like molecules like DNA and RNA, Krylov's group made computer models of paired, ring-shaped uracil molecules, and investigated what might happen to these doubled forms (dimers) when they were subjected to ionization – the removal of one or more electrons with resulting net positive charge.

Uracil is one of the four nucleobases of RNA, whose structure is similar to DNA except that, while both use the bases adenine, cytosine, and guanine, in DNA the fourth base is thymine and in RNA it's uracil. The USC group used a uracil dimer labeled 1,3-dimethyluracil – "a strange creature that doesn't necessarily exist in nature," says CSD's Amir Golan, who led the Berkeley Lab team at the ALS. The purpose of this strange creature, Golan says, is to block hydrogen bonding of the two identical monomers of the uracil dimer by attaching a methyl group to each, "because methyl groups are poison to hydrogen bonds."

The uracils could still bond in the vertical direction by means of pi bonds, which are perpendicular to the usual plane of bonding among the flat rings of uracil and other nucleobases. "Pi stacking" is important in the configuration of DNA and RNA, in protein folding, and in other chemical structures as well, and pi stacking was what interested the USC researchers. They brought their theoretical calculations to Berkeley Lab for experimental testing at the ALS's Chemical Dynamics beamline 9.0.2.

To examine how the molecules were bonded, Golan and his colleagues first created a gaseous molecular beam of real methylated uracil monomers and dimers, then ionized them with a beam of energetic ultraviolet light from the ALS synchrotron. The resulting species were weighed in a mass spectrometer to see how the uracil had responded to the extra boost of energy.

"Uracils could be joined by hydrogen bonds or by pi bonds, but these uracils had been methylated to block hydrogen bonds. So what we expected to see when we ionized them was that if they were bonded, they would have to be stacked on top of each other," Golan says. Instead of holding together by pi bonds, however, when ionized some uracil dimers had fallen apart into monomers that carried an extra proton.

Where the protons come from

"What we did not expect to see was proton transfer," Golan says. "Surprising as this was, we needed to find where the protons were coming from. The methyl groups consist of a single carbon atom and three hydrogen atoms, but methylated uracil has other hydrogens too. Still, the methyl groups were the natural suspects."

To test this hypothesis, the researchers invited colleagues from Berkeley Lab's Molecular Foundry to join the collaboration. They created methyl groups in which the hydrogen atoms – which like most hydrogen had single protons as their nuclei – were replaced by deuterium atoms, "heavy hydrogen" atoms with nuclei consisting of a proton and a neutron of virtually the same mass.

The molecular beam experiment was repeated at the ALS, and once again some of the methylated uracil dimers fell apart into monomers upon ionization. This time, however, the tell-tale monomers were not simply protonated, they were deuterated.

Says Golan, "By looking at the mass of the fragments we could see that instead of uracil plus one" – the mass of a single proton – "they were uracil plus two" – a proton and neutron, or deuteron. "This proved that indeed the transferred protons came from the methyl groups."

The experiment showed that proton transfer in this case followed a very different route from the usual process of hydrogen bonding. Here the transfer involved not just an attraction between molecular arrangements that were slightly positively charged and others that were slightly negatively charged, as in a hydrogen bond. Instead it required significant rearrangements of the two uracil dimer fragments, to allow protons of hydrogen atoms in the methyl group on one monomer to move closer to an oxygen atom in the other. Theoretical calculations of the new pathway were led by USC's Krylov and Ksenia Bravaya.

The moral of the story, says Golan, is that methyl groups do not always kill proton transfer. "Granted, this was a model system – what we did was ionize the uracil systems in the gas phase instead of in solution, as would be the case in a living organism," he says. "Nevertheless, we showed that proton transfer is possible without hydrogen-bonding networks. Which means there could be unsuspected pathways for proton transfer in RNA and DNA and other biological processes – especially those that involve pi-stacking – as well as in environmental chemistry and in purely chemical processes like catalysis."

The next step: a range of new experiments to directly map proton transfer rates and gain structural insight into the transfer mechanism, with the goal of visualizing these unexpected new pathways for proton transfer.

More information: "Ionization of dimethyluracil dimers leads to facile proton transfer in the absence of H-bonds," by Amir Golan, Ksenia B. Bravaya, Romas Kudirka, Oleg Kostko, Stephen R. Leone, Anna I. Krylov, and Musahid Ahmed, is published by Nature Chemistry and appears in advance online publication at http://www.nature. … m/index.html .

Provided by Lawrence Berkeley National Laboratory (news : web)

Cyborg snail produces electricity

But whereas the grapes and cockroaches could generate electricity for just days or weeks, Evgeny Katz, a professor of chemistry at Clarkson University in Potsdam, New York, and colleagues have shown that the snail can generate electricity for many months at a time. And in spite of the electrodes in their shells, the snails live long, healthy lives.

“The animals are quite fit - they eat, drink and crawl,” Katz told Nature News. "We take care to keep them alive and happy.”

Although a snail's tissues and organs are bathed in blood, or haemolymph, it takes time to regenerate its glucose levels, which means snails don't generate very large amounts of power. For the first few minutes, the researchers could extract 7.45 microwatts, but this power decreased to just 0.16 microwatts during long-term, continuous extraction. The main cause of this decay comes from the local depletion of glucose at the electrode surface. Still, the snail's eating and resting could sufficiently regenerate its overall glucose levels, allowing it to “recharge” and produce sustainable electrical power.

These snails - as well as other potential electrified creatures such as worms and insects - could be useful for powering low-power devices, such as sensors and wireless transmitters. The US Department of Defense is funding cyborg research in the hopes of creating bugs that can gather information about their environment while crawling around. Researchers are also investigating medical applications, in which a patient's implantable biofuel cell could use his or her own blood glucose to power medical devices such as pacemakers.

In the future, the researchers at Clarkson University plan to electrify lobsters in the same way as the snails, with the hopes that the larger animals' metabolism could provide more power.

More information: Lenka Halámková, et al. "Implanted Biofuel Cell Operating in a Living Snail." Journal of the American Chemical Society. DOI: 10.1021/ja211714w

Abstract
Implantable biofuel cells have been suggested as sustainable micropower sources operating in living organisms, but such bioelectronic systems are still exotic and very challenging to design. Very few examples of abiotic and enzyme-based biofuel cells operating in animals in vivo have been reported. Implantation of biocatalytic electrodes and extraction of electrical power from small living creatures is even more difficult and has not been achieved to date. Here we report on the first implanted biofuel cell continuously operating in a snail and producing electrical power over a long period of time using physiologically produced glucose as a fuel. The “electrified” snail, being a biotechnological living “device”, was able to regenerate glucose consumed by biocatalytic electrodes, upon appropriate feeding and relaxing, and then produce a new “portion” of electrical energy. The snail with the implanted biofuel cell will be able to operate in a natural environment, producing sustainable electrical micropower for activating various bioelectronic devices.

 

Researcher sees marine nutraceuticals as growth industry

Lee, a professor emeritus of food sciences, describes nutraceuticals as a cross between pharmaceuticals and nutrition, something that "provides health benefits above and beyond traditional nutrients. The nutraceutical market is dominated by terrestrial sources, like cranberries that provide antioxidants. Marine nutraceuticals are something new, and now it is getting a lot more attention," he said.

The URI food scientist says that the "big ticket item" among marine nutraceuticals is fish oil, which contains omega 3 fatty acids that provide cardiovascular disease risk reductions, immune function improvements, brain health, and reductions in inflammation from rheumatoid arthritis. Most fish oil products are derived from anchovies and sardines caught in the waters off Peru and Chile.

In Rhode Island, seaweed could play a role in the nutraceutical industry, as some varieties are a source of compounds beneficial to human and animal health. One species of seaweed called rockweed (Ascopyllum nodosum) that Lee is studying is abundant along the coast of New England and is already being used as an agricultural fertilizer and as an additive to animal feeds. Lee says it also has bioactive components that are useful in managing weight, lowering cholesterol and slowing the digestion of sugars and carbohydrates.

Lee's former postdoctoral student, Emmanouil Apostolidis, is studying the seasonal variation of these beneficial properties to determine the best time of year to harvest the seaweed and examining its stability to determine how long it can remain on the shelf before it's potency declines."

Lee is also working with squid processors in North Kingstown and Point Judith in the development of nutraceuticals from the by-products of squid processing. A global pet food company has already been in touch with Lee about using squid by-products in its products to improve animal health.

In addition, Lee is collaborating with the Rhode Island Commercial Fisheries Research Foundation and local scallop fishermen on research to find a beneficial use for the by-products of scallop harvesting.

"We only consume the adductor muscle of scallops, and the rest – 70 percent of it – is thrown overboard," said Lee. "We're investigating its potential. One of my assistants, Bouhee Kang, is working on this project. It may stimulate digestion enzyme activity and help people who have difficulty digesting oily foods. There is a big market for digestion relief supplements, especially in Asia."

Lee believes that Rhode Island could benefit from more research into marine nutraceuticals.

"In the past, most of the funding for this kind of research in the U.S. has gone to the development of drugs from marine organisms. I hope the funding will come soon for nutraceuticals as well," said Lee, who recently organized an international symposium on global trends in marine nutraceuticals. "It has great potential, and it could give a big boost to the Rhode Island economy."

Provided by University of Rhode Island (news : web)